Peripheral tolerance prevents self-reactive T cells that escape thymic negative selection from causing autoimmunity. In the prevailing model of intrinsic peripheral tolerance, quiescent tissue-resident dendritic cells (DC) migrate to lymph nodes (LN) where they cross present self-Ag to T cells, which then undergo deletion or anergy. We have shown that CD8 T cell tolerance to an epitope derived from tyrosinase, a protein whose expression is normally confined to melanocytes and retinal pigment epithelial cells, is not mediated by either thymic negative selection or cross-tolerance. Rather, CD8 T cells undergo abortive proliferation and deletion mediated by LN resident lymphatic endothelial cells (LEC) that adventitiously express tyrosinase. We and other groups have established that at least two other LN stromal subsets can also induce CD8 T cell tolerance. These subpopulations each express distinct subsets of many peripheral tissue Ags, suggesting that they represent a major, previously unrecognized mechanism of systemic intrinsic peripheral tolerance. The goal of the present application is to provide evidence to support this hypothesis, with a specific focus on LEC, and to explore factors that influence the ability of LEC to carry out this function. In Aim 1 we will use a newly developed model system to determine the mechanisms by which LEC, which express MHC-II molecules, tolerize CD4 T cells, and explore the role of antigen expression levels on tolerogenic outcome in both CD4 and CD8 T cells. In Aim 2 we will explore the significance of PD-L1, which LEC express at a higher level than any other LN cell population, on tolerance inducedby different antigen presenting cells. Our preliminary studies suggest that PD-L1 signaling can occur without simultaneous Ag engagement, and we hypothesize that LEC act as gatekeepers to enforce the quality of immune responses. We will also establish the mechanisms by which proliferation of T cells destined to undergo deletion is induced, and the role of LEC in this process. Proliferation appears to be an essential element of the deletion process, but also can enable accumulation of autoreactive T cells. In Aim 3 we will define the mechanisms by which LEC that reside in LN, as opposed to those in tissue lymphatic vessels, are able to induce tolerance based on their expression of peripheral tissue Ags and a set of immunomodulatory molecules, including PD-L1. We will define the cellular and molecular components that influence expression of these molecules in LN resident LEC, and use gene expression analyses to identify the transcription factors that control expression of these molecules and peripheral tissue Ags in LEC. Collectively, we expect the studies supported by this grant to lead to significant new insights into the mechanisms by which peripheral self-tolerance is established and maintained, which will be relevant in understand the development of autoimmune disease and the ways in which self-tolerance limits immune responses to cancer cell Ags that are derived from tissue specific proteins.